All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
After vascular injury, platelets rapidly adhere to damaged blood vessels and trigger a complex cascade of events that result in thrombus formation. The demand lot platelet transfusions has continued to increase during the last several decades51. Platelets can only be stored for less than a week, creating a continuous challenge for donor dependent programs. Shortages in the supply of platelets can have potentially life-threatening consequences, especially in patients where multiple transfusions are necessary. Repeated transfusions may also lead to refractory responses that are linked to immunity mediated host reaction and may require costly patient matching52;53. The ability to generate patient-matched platelets in vitro would provide significant advantages in these clinical scenarios.
Limitations in the supply of platelets can have potentially life-threatening consequences for transfusion-dependent patients with unusual/rare blood types, particularly those who are alloimmunized, and patients with cancer or leukemia who, as often happens, develop platelet alloimmunity. Frequent transfusion of platelets is clinically necessary because the half-life of transfused human platelets is 4-5 days. Moreover, platelets from volunteer donor program are at the constant risk of contaminations of various pathogens. Platelets cannot be stored frozen, thus the ability to generate platelets in vitro would provide significant advances for platelet replacement therapy in clinical settings. For more than a decade, human hematopoietic stem cells (HSC, CD34+) from bone marrow (BM), cord blood (CB) or peripheral blood (PB) have been studied for megakaryocyte (MK) and platelet generation. With the combinations of cytokines, growth factors and/or stromal feeder cells, functional platelets have been produced from HSCs with significant success1;2. However, HSCs are still from donors and have limited expansion capacity under current culture conditions, which likely prevent the large-scale production and future clinical applications.
Human embryonic stem cells (hESC) can be propagated and expanded in vitro indefinitely, providing a potentially inexhaustible and donorless source of cells for human therapy. Differentiation of hESCs into hematopoietic cells has been extensively investigated in vitro for the past decade. The directed hematopoietic differentiation of hESCs has been successfully achieved in vitro by means of two different types of culture systems. One of these employs co-cultures of hESCs with stromal feeder cells, in serum-containing medium3:4. The second type of procedure employs suspension culture conditions in ultra-low cell binding plates, in the presence of cytokines with/without serum5-7; its endpoint is the formation of EBs. Hematopoietic precursors as well as mature, functional progenies representing erythroid, myeloid, macrophage, megakaryocytic and lymphoid lineages have been identified in both of the above differentiating hESC culture systems3-6:8-14. Previous studies also generated megakaryocytes/platelets from hESCs by co-culturing with stromal cells in the presence of serum15;16. Platelets derived from hESCs possess the potential for transfusion medicine purposes if they can be generated efficiently and in large scale. More importantly, platelets do not have a nucleus and contain only minimal genetic material, and can be irradiated before transfusion to effectively eliminate any contaminating cells, such as an undifferentiated hESC. Therefore, safety should not be an issue. However, the yield of megakaryocytes/platelets in the above studies was low15;16. There remains a need in the art for efficient and controlled differentiation of hESCs into homogeneous megakaryocytic populations and subsequent functional platelets.